The present invention relates to novel variants of human hypoxia-inducible factor (HIF)-1xcex1, as well as to the use of the said variants in screening assays for the elucidation of functional motifs in HIF-1xcex1 and for the identification of compounds that modulate the function of HIF-1xcex1, said compounds being potentially useful in the regulation of target genes normally associated with HIF-1xcex1 such as genes involved in angiogenesis, erythropoiesis, and glycolysis.
Oxygen plays a critical biological role as the terminal electron acceptor in the mitochondria of vertebrate cells. During evolution, these cells have developed ways to sense alterations in oxygen levels and, during this process, acquired the ability to conditionally modulate the expression of genes involved in adaptive physiological responses to hypoxia including angiogenesis, erythropoiesis, and glycolysis. These genes include vascular endothelial growth factor, eryhtropoietin, several glycolytic enzymes and inducible nitric oxide synthase, and have all been shown to contain hypoxia responsive elements (HREs) (for reviews, see Guillemin and Krasnow (1997) Cell 89, 9-12; Wenger and Gassmann (1997) Biol. Chem. 378, 609-616). Under hypoxic conditions these response elements are recognized by a heterodimeric complex consisting of the hypoxia inducible factor (HIF)-1xcex1 and Arnt (Wang et al. (1995) Proc. Natl. Acad. Sci. USA 92, 5510-5514; Gradin et al. (1996) Mol. Cell. Biol. 16, 5221-5231). Both these transcription factors belong to the rapidly growing family of basic-helix-loop-helix (bHLH)-PAS (Per, Arnt, Sim) proteins. bHLH/PAS transcription factors play diverse biological roles.
The recent generation of HIF-1xcex1- and Arnt-deficient embryonal stem cells and mice have indicated critical roles of both these factors in cardiovascular development and regulation of HRE-driven target genes (Maltepe et al. (1997) Nature 386, 403-407). The mechanism of hypoxia-dependent formation and activation of the HIF-1xcex1-Arnt complex is presently poorly understood. The present inventors and others have recently demonstrated that HIF-1xcex1 protein levels are specifically and massively upregulated under hypoxic conditions in most if not in all cells. Since HIF-1xcex1 mRNA levels are unaltered in response to hypoxia, this mode of regulation appears to occur via a post-transcriptional step involving stabilization of HIF-1xcex1 protein levels (Huang et al. (1996) J. Biol. Chem. 271, 32253-32259; Kallio et al. (1997) Proc. Natl. Acad. Sci. USA 94, 5667-5672; Salceda and Caro (1997) J. Biol. Chem. 272, 22642-22647) preceding recruitment of Arnt and generation of a nuclear DNA binding complex.
Import of transcription factors into the nucleus is frequently a conditionally regulated process that occurs in response to various external and internal stimuli as well as to a developmental cues (Vandromme et al. (1996) Trends Biochem. Sci. 21, 59-64). Thus, this process can constitute a critical mechanism of regulation of transcription factor activity. Active, energy-dependent transport of proteins to the nucleus requires the presence of one or several nuclear localization signals (NLSs) within the transported protein (or its interaction partner). NLS motifs are short amino acid moieties that can be divided into two main groups: (i) the SV40 large T antigen type of NLS, characterized by a single cluster of four or more consecutive basic residues; and (ii) a bipartite NLS, consisting of two basic residues, a spacer of any ten amino acids, and a basic cluster where three of the next five residues are basic (Vandromme et al., supra, and references therein).
Purified HIF-1xcex1, its amino acid sequence and polynucleotide sequence are disclosed by Wang et al. (1995) Proc. Natl. Acad. Sci. USA 92, 5510-5514 and in WO 96/39426. It has been suggested (Jiang et al. (1997) J. Biol. Chem. 272, 19253-19260) that amino acids 531-826 of HIF-1xcex1 contain two transactivation domains, TAD-N (amino acids 531-575) and TAD-C (amino acids 786-826). Further, Pugh et al. ((1997) J. Biol. Chem. 272, 11205-11214) defined two minimal domains within HIF-1xcex1 (amino acids 549-582 and 775-826) each of which could act to confer transcriptional activation.
There is a need for novel variants of human HIF-1xcex1, which variants can be used in screening assays for the elucidation of functional motifs in HIF-1xcex1 and for the identification of compounds that modulate the function of HIF-1xcex1, said compounds being potentially useful in the regulation of target genes normally associated with HIF-1xcex1 such as genes involved in angiogenesis, erythropoiesis, and glycolysis.